Fermentation Optimization And Strain Improvement For 2,3-Butanediol Production Utilizing Cheese Whey

2,3-butanediol is a kind of important chemical raw material,widely used in many fields,such as chemical industry,food,fuel and aerospace industry.Cheese whey is rather cheap and production of 2,3-butanediol with cheese whey as substrate by biological process is very attractive in 2,3-butanediol production.In previous study,the concentration and productivity of 2,3-butanediol with cheese whey(powder)as substrate are not satisfactory.In this study,the related process of 2,3-butanediol production from cheese whey was studied to produce 2,3-butanediol from cheese whey efficiently,with the purpose of providing a reference for industrialization of 2,3-butanediol production from cheese whey.In fermentation of 2,3-butanediol,other byproducts are produced,which decrease the yield of 2,3-butanediol.In this study,with metabolic engineering,the ethanol metabolic pathway of the relevant strain was modified to increase the 2,3-butanediol production capacity.(1)By comparing the fermentation result of 2,3-butanediol utilizing lactose,the capacity of producing 2,3-butanediol of strains Klebsiella pneumoniae CICC 10781 and Klebsiella oxytoca CICC 21518 was found higher than other 3 strains.By further comparing the fermetation result of these two strains with cheese whey,lactose,glucose,galactose,the mixture of glucose and galactose as substrates,and considering the safety of strains,the strains CICC 21518 was chosen as starting strains.(2)The fermentation conditions with cheese whey as substrate were optimized by flask experiment and the optimized conditions were initial pH of 6.5,rotation speed of 180 r/min,temperature of 32 ?,liquid volume of 125 mL and inoculation amount of 7%.On the basis of optimized fermentation conditions,the culture medium was optimized and the optimized medium were:cheese whey 110 g/L,urea 0.54 g/L,sodium citrate 3.65 g/L,sodium acetate 5.45 g/L,K2HPO4 5g/L,Mn2+ 0.025 g/L,Mg2+0.05g/L,Zn2+ 0.005 g/L and Fe2+ 0.005 g/L.(3)In fermentor experiment,the fermentation control strategy with cheese whey as substrate was established initially.The two-stage pH control strategy was optimized for 2,3-butanediol production:from 0 h to 8 h,the pH was kept at 6.5 and from 8 h to the ending of fermentation,the pH was kept at 6.The optimized stirring speed was 500 r/min and the 2,3-butanediol concentration was further increased by fed-batch fermentation.At last,the fermentation ended at 60 h,and the final concentration,yield,and productivity of 2,3-butanediol were 57.63 g/L,0.397 g/g and 0.961 g/(L·h),respectively.The final concentration and productivity of 2,3-butanendiol was largely increased than that in previous study.(4)To decrease the production of ethenol,the adhE gene of Klebsiella oxytoca CICC 21518 was knocked out.The ethanol concentration was decreased by 93%and 2,3-butanediol concentration was increased by 8.3%.